Cytokine upsurge among drug-resistant tuberculosis endorse the signatures of hyper inflammation and disease severity

Tuberculosis (TB) elimination is possible with the discovery of accurate biomarkers that define the stages of infection. Drug-resistant TB impair the current treatment strategies and worsen the unfavourable outcomes. The knowledge on host immune responses between drug-sensitive and drug-resistant infection is inadequate to understand the pathophysiological differences and disease severity. The secreted proteins, cytokines display versatile behaviour upon infection with Mycobacterium tuberculosis (MTB) and their imbalances often tend to assist disease pathology than protection. Therefore, studying these soluble proteins across TB infection spectrum (drug-resistant TB, drug-sensitive TB, and latent TB) may unveil the disease mediated responses and unique stage specific cytokine signatures. Thus, we sought to determine the plasma cytokine levels from healthy, latently infected, drug-sensitive, and drug-resistant TB individuals. Our study revealed top 8 cytokines (IL-17, IL-1α, IL-2, IL-10, IL-5, IFN-γ, TNF-α and IL-6) and their biomarker abilities to discriminate different stages of infection.

Study population and design. The study comprised of healthy controls (HC) (n = 40), latently infected individuals (LTB) (n = 40), drug-sensitive TB (DS-TB) (n = 40) and drug-resistant TB (DR-TB) (n = 40). Quan-tiFERON-Interferon Gamma Release Assay (IGRA) was performed for the healthy individuals who are asymptomatic for TB. IGRA negative individuals are assigned as HC and IGRA positive were taken as LTB positive individuals. DS-TB and DR-TB infected participants were recruited from the corporation centres in and around Chennai based on their routine TB diagnosis. Participants diagnosed with TB by smear or culture or GeneXpert and sensitive for first line anti-TB drugs are considered as DS-TB. DR-TB participants are those who are diagnosed with TB along with resistance to rifampicin alone or isoniazid alone or combination of both (either primary or acquired resistance) that was confirmed through GeneXpert or line probe assay (LPA) or drug sensitivity test (DST). Our study, has clinically well characterized cohort which has been diagnosed for TB, exclusive of other infections and other co-morbid conditions like Diabetes Mellitus, HIV, HCV and HBV. Blood was collected at one time point from DS-TB/DR-TB groups before the treatment initiation. Plasma was separated by centrifuging blood at 2600 rpm for 10 min and stored at − 80 °C until further analysis.
Statistical analysis. Graph-Pad PRISM Version 9.0 (GraphPad Software, CA, UA) was used to analyse the statistical difference among the groups. R software version 4.2.0 (R Core Team, 2022) was used to perform random forest analysis and principal component analysis. Shapiro-Wilk normality test was performed to test the normality of the data. Cytokine concentrations are shown as median and interquartile range (IQR) and demographic characteristics as numbers and percentages. Demographic characteristics were assessed using Fishers Exact test at 5% level of significance. Statistical significance between the study groups (DR-TB, DS-TB, LTB, and HC) for hematology and cytokine observations were analysed using Dunn test corrected for multiple comparisons using Bonferroni test. The sensitivity and specificity were assessed using receiver operating characteristic curve (ROC) analysis. The importance of the cytokines was ranked through random forest (RF) analysis. The dimensionality reduction was carried out using principal component analysis (PCA) to identify the classification pattern of the ranked cytokines. Spearman correlations were carried out to understand the relationship between cytokines. Hierarchical clustering was performed to visualize the segmentation of these cytokines in the study groups using SOM module in Multi Experiment Viewer Application (http:// www. tm4. org/). p < 0.05 was considered statistically significant.

Results
Basic characteristics. We carried out the immunological assays on plasma samples of 160 individuals.
Of these 160 individuals, 40 individuals with drug-resistant Tuberculosis (DR-TB), 40 individuals with drugsensitive tuberculosis (DS-TB), 40 individuals with latent tuberculosis (LTB) and 40 healthy control (HC) individuals. Further demographics and haematological data of participants are described in Table 1 www.nature.com/scientificreports/ differences were observed in the hematological parameters except RBC, eosinophil and basophil count were mentioned in Table 1 with their respective p values.
LTB individuals exhibited significantly increased levels of IFN-γ (p = 0.0119), IL-2 (p < 0.0001), TNF-α (p = 0.0347) and IL-17 (p = 0.0001) in comparison to HC group. Thus, the clinical spectrum of TB disease/ infection is associated with altered levels of cytokines. in the cytokine expression profile were assessed by hierarchical clustering using log 2 transformed and mean normalized values. As shown in Fig. 2, heat map reveals the changes in the cytokine trend from latency to drug sensitive and to drug resistance with a greater number of cytokine accumulation during severe condition like DR-TB. Prior to infection, the disease protective latent condition presented the cytokine panel with mild or moderate levels of TNF-α, IFN-γ, IL-2, IL-17, and IL-6. In the diseased state, DS-TB individuals presented differential cytokine expression with high levels of IFN-γ, IL-2, IL-17, and IL-6; moderate levels of IL-1α, IL-12p70, TNF-α and IL-10 and mild levels of GM-CSF. DR-TB individuals are associated with abundant cytokine expression of TNF-α, IFN-γ, IL-2, IL-17, IL-6, and IL-10 and with moderate expression of GM-CSF, IL-12p70, IL-1α and IL-1β. As per the heat map, the expression of TNF-α, IL-6 and IL-10 are abundantly increased in DR-TB whereas, IL-1α expression is higher in DS-TB compared to other groups. On contrary, IL-5 expression is moderate in the LTB and HC group and low in DS-TB and DR-TB groups. Thus, these analyses help to reveal the power of cytokines to demarcate the spectrum of TB disease/infection (DR-TB, DS-TB, and LTB) from HC.
Diagnostic performance of top 8 cytokines for bifurcation of DR-TB, DS-TB, LTB, and HC. We conducted a ROC analysis to determine the diagnostic capabilities of each cytokine to distinguish between the groups of study. The representative curves showing the cytokines with the best diagnostic accuracy between and among these groups are shown in Fig. 3. IL-17 exhibited AUC = 0.97 and significantly (p < 0.0001) discriminate DR-TB from DS-TB (Fig. 3c). In addition, we performed a random forest (RF) analysis to understand the importance of these cytokines and their discrimination toward the separation of study groups. According to the order of importance, RF plots of overall comparison (HC vs LTB vs DS-TB vs DR-TB) presented IL-17, IL-1α, IL-2, IL-10, IL-5, IFN-γ, TNF-α and IL-6 as the topmost classifiers (Fig. 4a). This was in accordance with the ROC results where these cytokines displayed higher AUC values of above 0.8. Similarly in the subgroup comparisons, the same IL-17 was obtained as the topmost classifier for HC vs LTB/DS-TB/DR-TB ( Fig. 5a-1,a-3) and DS-TB vs DR-TB (Fig. 5a-6) whereas, IL-1α for LTB vs DS-TB (Fig. 5a-4) and IL-10 for LTB vs DR-TB (Fig. 5a-5).

Discussion
TB biomarkers can be considered crucial to achieve the global TB elimination targets. The effort to understand causal factors like cytokines and their differential expression during different stages of TB is valuable in identifying unique biological signatures 22 . The phenomena of host bodily functions are activated, shaped, and responded through multifunctional cytokines. However, adequate balance of cytokines is cardinal for the appropriate protective responses 20 as the hypo or hyper responses is often dangerous and progress the disease severity. The essence of balanced production of pro-(IL-17) and anti-inflammatory cytokines (IL-10) for MTB clearance was apparent from the experiment with macaques 17 . The diagnostic utility of the cytokines is proven with QFT-IGRA for MTB infection, but less sensitive to discriminate LTB from active TB [22][23][24] . Therefore, approaches with cytokine blend grab attention to improve their diagnostic abilities. Considering this, we attempted to estimate an array of cytokines (14 plex) during latency, drug-sensitive and drug-resistant conditions compared to normal healthy condition. The inclusion of all the stages of TB disease spectrum aids an advantage over previous studies that either lack LTB 25,26 or DR-TB 8,22 in revealing the serial increase of cytokines from LTB to DS-TB and DR-TB. This trend marks the hyper immune responses and severity of DR-TB by the accumulation of multiple cytokines (IL-12p70, TNF-α, INF-γ, IL-2, IL-17, IL-6, and IL-10) at higher concentration. Furthermore, the numbers and the levels were tapered in DS-TB (INF-γ, IL-2, IL-17, and IL-6) with a further reduction in LTB (INF-γ, IL-2, and IL-17). The possible reason for the higher IFN-γ levels in DR-TB from our study is due to the IFN-γ demand in macrophages to kill the drug resistant mycobacteria. In addition, the elevated IL-10 levels suggest the loss of balance between pro-and anti-inflammatory cytokines that could cause immune suppression. Similar to our findings, DR-TB groups were previously suggested with increased TNF, IFN-γ and IL-10 26,27 associated with marked necrosis and resistance to drugs. Mensah et al. www.nature.com/scientificreports/ stated no differences between the DS-TB and DR-TB with smaller sample size 25 . Though the studies for DR-TB is minimal and with varying cytokine levels, the obtained differences from the current study offer a classical view of better separation of DR-TB from DS-TB, LTB, and HC. All nucleated cells produce cytokines with either antagonistic or synergistic effects 11 depends on the infection milieu and the host-pathogen interaction. The release of Th1 and Th17 inflammatory cytokines (IL-2,  www.nature.com/scientificreports/ TNF-α, IFN-γ, IL-12 and IL-17) are correlated with host protective responses against TB infection 27,28 and that was in connection with MTB clearance and sterilization of granulomas in macaques 17 . The so-called protective cytokines switch to pathological responses due to their overproduction and loss of balance in DR-TB and DS-TB from our study as stated earlier by Kumar et al., 2019 where the type 1 and 17 cytokines assist disease pathology in PTB 8 . The inflammation driven by monocytes are sceptical for pathological response in TB as their partial depletion in patients with chagas disease lessened the pro-(IFN-γ, IL-2 and IL-5) and anti-inflammatory (IL-10) cytokine levels with improved antigen presentation ability of B cells 30 . Peripheral accumulation of monocytes, as evidenced through increased MLR could be the major source of the driving systemic inflammation and, elevating the circulatory cytokine levels among the DR-TB, when compared to the effector T cells. The damage caused by DR-TB is quite long term as the heightened levels of TNF, IFN-γ and IL-12 were sustained even after ATT in DR-TB individuals than DS-TB 31 . Cytokines work in a cascade fashion during TB, where IL-12 controls the Th1 cytokine (IL-2/IFN-γ) production 8,13,32 . The IFN-γ in turn activates macrophages 26,33 that induce TNF-α secretion for infection containment and mycobacterial growth restriction 26,34,35 . TNF-α and IL-1α are the predominant contributors of granuloma formation 22,36 . However, their protective effect was down regulated by the high IL-6/IL-10 levels in association with pSTAT3/SOCS3 expression which ultimately led to immune suppression and impaired T cell function 37 . In addition, type 1 cytokines (IFN-γ and TNF-α) determines the degree of infection by their discrete association with bacterial density 8 and turns their personality of protection towards disease dissemination. Cytokines were extensively investigated during TB disease to understand their association with bacterial burden (IL-17A, IFN-γ, TNF-α and IL-6) 8,38 , cavitation (IL-1β) 19 , disease severity and pathogenesis (IL-17A, IL-1β, and IL-6) 8,9 , and time to culture conversion (IL-17A) 8 . The ideal focus on cytokine patterns determines their biomarker abilities for prognosis, TB detection (IL-2, IFN-γ and TNF-α) 39,40 , discrimination of TB from LTB (TNF, IL-12p40 and IL-17) 41 and treatment outcome (IL-6, IL-1β and IFN-γ /IL-10 ratio) 38,42,43 . However, the differences reported were varied between the studies and was complexed to find the true candidates. Smaller sample size might mask the true reflection of the disease status as that was apparent from our previous experience with cytokines that contrasted the current observation 44 . Dimensionality reduction of the current data enabled   c. d. www.nature.com/scientificreports/ top 8 cytokines (IL-17, IL-1α, IL-2, IL-10, IL-5, IFN-γ, TNF-α and IL-6) with better separation of 4 study groups in different clusters with the accuracy of 94.4%. Moreover, further reduction to the topmost classifiers for DR-TB vs DS-TB (IL-17, IL-5 and TNF-α), DS-TB vs LTB (IL-1α, IL-5 and IL-10) and LTB vs HC (IL-17, IL-2 and IFNγ) may account for 100% accuracy in discriminating between them. Though systemic responses don't reflect the accurate in-vivo status of granuloma 17 , the observations from the current study gives an overview of cytokine behaviour across TB disease spectrum. Since cytokines are multifunctional, the combined effect of cytokines and their signalling network at disease setting must be explored further both in-vitro and in-vivo to understand their intrinsic role in TB pathogenesis. Our study has limitation, that the details of culture conversion and the treatment outcome is unknown. However, our study has advantage over sample size, inclusion, and comparison of TB disease spectrum (DR-TB, DS-TB, and LTB) with healthy controls (HC).
In conclusion, IL-17 exhibited stage specific increase with area under curve value above 0.9 that decipher good sensitivity and specificity across the infection spectrum. Our findings could identify stage specific cytokines, particularly upsurge of specific cytokines was found in DR-TB exhibiting hyper immune responses and disease severity. Future validation of these cytokine signatures in larger cohort at multiple sites may uncover their biomarker potency and their role in host immune system towards drug-resistance.

Data availability
The data supporting the findings of this article will be made available by the corresponding author, upon request.